Soluble ErbB3 and treatment of cancer

ABSTRACT

Regulation of sErbB3 isoforms in methods of regulating heregulin activity or ErbB receptor activities is disclosed. Cancer therapeutics and methods of therapeutically treating cancer comprising sErbB3 are also disclosed. Detection of sErbB3 in biological samples for risk assessment and prevention, screening, diagnosis, prognosis, theragnosis, evaluation of responsiveness to treatment, and/or monitoring of disease progression, recurrence, or metastasis of a cancer is disclosed as well. In examples, sErbB3 nucleic acid sequences, polypeptides, molecular probes, and antibodies are useful agents for regulation, expression, detection, and cancer therapeutics related to sErbB3.

This application is a continuation-in-part of U.S. application Ser. No.10/159,353 filed May 31, 2002, which claims priority to U.S. ProvisionalApplication 60/294,824 filed May 31, 2001.

The disclosed invention was made with the support of a grant from theNational Cancer Institute (CA85133). The United States Government hascertain rights in the invention.

FIELD OF INVENTION

Embodiments of the present invention generally pertain to methods andtherapeutics that relate to soluble ErbB3 proteins (sErbB3), such asp85-sErbB3, p45-sErbB3 and other isoforms of sErbB3. Embodiments of thepresent invention pertain to methods of treating cancer and cancertherapeutics comprising an sErbB3 or an sErbB3 agent regulatingexpression or activity of an sErbB3. Additional embodiments of thepresent invention pertain to sErbB3 regulation of heregulin or at leastone ErbB receptor activity and treatment of associated conditions. Otherembodiments relate to risk assessment and cancer prevention, screening,diagnosis, prognosis, theragnosis, monitoring of responsiveness totreatment, and monitoring of disease progression, recurrence, ormetastasis of a cancer based on aberrant sErbB3 concentrations orlocalization in biological samples, for example serum or tissue.

BACKGROUND

The following background information is provided to assist the reader tounderstand the invention disclosed below and the environment in which itwill typically be used. The terms used herein are not intended to belimited to any particular narrow interpretation unless clearly statedotherwise, either expressly or impliedly, in this document.

The heregulins (also called neuregulins, neu differentiation factor(NDF), acetylcholine receptor inducing activity (ARIA), and glial growthfactors (GGFs)) are a family of growth factors that activate members ofthe ErbB/EGF receptor family (Holmes, Sliwkowski et al. 1992; Peles,Bacus et al. 1992; Wen, Peles et al. 1992; Falls, Rosen et al. 1993;Marchionni, Goodearl et al. 1993). Isoforms of heregulins, all of whicharise from splice variants of a single gene, NRG-1 (neuregulin-1), havebeen cloned and classified into the α and β subgroups based onstructural differences in their epidermal growth factor (EGF) likedomains (Holmes, Sliwkowski et al. 1992).

ErbB-mediated signal transduction exerted by heregulins has beenimplicated in the regulation of diverse biological events includingSchwann cell differentiation, neural regulation of skeletal muscledifferentiation, heart development, and proliferation anddifferentiation of normal and malignant breast epithelial cells (Alroyand Yarden 1997; Sundaresan, Penuel et al. 1999). Research has shownthat breast carcinoma cells respond to heregulin by activating signaltransduction pathways that result in cellular proliferation,differentiation, as well as morphogenesis. Carcinoma cells expressingheregulin are typically hormone-independent and associated with theability for metastasis in experimental studies.

ErbB3 is a transmembrane glycoprotein encoded by the c-erbB3 gene(Kraus, Issing et al. 1989; Plowman, Whitney et al. 1990). The ErbB3receptor belongs to the ErbB family which is composed of four growthfactor receptor tyrosine kinases, known as ErbB1/EGFR/HER1,ErbB2/Neu/HER2, ErbB4/HER4, as well as ErbB3/HER3. ErbB3 and ErbB4 arereceptors for heregulins, whereas ErbB2 is a coreceptor (Carraway andBurden 1995). These receptors are structurally related and include threefunctional domains: an extracellular ligand-binding domain, atransmembrane domain, and a cytoplasmic tyrosine kinase domain (Plowman,Culouscou et al. 1993). The extracellular domain can be further dividedinto four subdomains (I-IV), including two cysteine-rich regions (II andIV) and two flanking regions (I and III). ErbB3 is unusual among thesereceptor tyrosine kinases in that its catalytic domain is defective.Despite its lack of intrinsic catalytic activity, ErbB3 is an importantmediator of both heregulin and epidermal growth factor (EGF)responsiveness by way of its ability to heterodimerize with other ErbBfamily members, but especially with ErbB2. Heregulin binding inducesErbB3 to associate with other members of the ErbB family to formheterodimeric receptor complexes. ErbB3 then activates the kinase of itspartner receptor which initiates a variety of c signaling cascades.ErbB3 also can relay information in response to diverse ligands, such asinterferon and TNF-alpha, presumably through receptor cross-talk.

The ErbB3 receptor is important in regulating normal and aberrantcellular growth and differentiation, metastasis, and relatedpathologies. Transgenic mice that have been engineered to overexpressheregulin in mammary glands have been reported to exhibit persistentterminal end buds and, over time, to develop mammary adenocarcinomas(Krane and Leder 1996). ErbB3 expression studies on tumor tissues and oncancer cell lines show frequent co-expression of both ErbB2 and ErbB3receptors (Alimandi, Heidaran et al. 1995; Meyer and Birchmeier 1995;Robinson, He et al. 1996; Siegel, Ryan et al. 1999). In addition,mammary tumors formed in transgenic mice that harbor an activated formof ErbB2 and metastasize to the lungs are associated with elevatedamounts of tyrosine-phosphorylated ErbB21Neu and ErbB3 (Siegel, Ryan etal. 1999). Many transformed cell lines used for experimental studies areeither estrogen-dependent (MCF-7 and T47D, the low ErbB2 expressers) orestrogen-independent (SKBR3, high ErbB2 expressers). However, these celllines do not exhibit metastatic phenotypes. When MCF-7 cells aretransfected to overexpress ErbB2, MCF-7 cells gain anestrogen-independent phenotype, however, they never metastasize. On theother hand, MCF-7 cells that overexpress heregulin gain a metastaticphenotype, suggesting that the heregulins play an active role inmetastasis (Hijazi, Thompson et al. 2000; Tsai, Hornby et al. 2000).

Five alternate ErbB3 transcripts arise from read-through of an intronand the use of alternative polyadenylation signals (Lee and Maihle 1998;Katoh, Yazaki et al. 1993). Using 3′-RACE four novel c-erbB-3 cDNAclones of 1.6, 1.7, 2.1, and 2.3 kb from a human ovariancarcinoma-derived cell line have been isolated (Lee and Maihle 1998).p85-sErbB3 is encoded by a 2.1 kb alternate c-erbB3 transcript (cDNAclone R31F) that is translated into a 543 aa protein composed ofsubdomains I through III and the first third of subdomain IV of theErbB3 extracellular domain, and a unique 24 amino acid carboxy-terminalsequence. p45-sErbB3 is encoded by a 1.7 kb alternate c-erbB3 transcript(cDNA clone R2F) that is translated into a 312 aa protein composed ofsubdomains I, II, and a portion of subdomain III of the extracellulardomain of ErbB-3 followed by two unique glycine residues. p50-sErbB3 isencoded by a 1.6 kb alternate c-erbB3 transcript (cDNA clone R1F) thatis translated into a 381 aa protein composed of subdomains I, II, and aportion of subdomain III of the extracellular domain of ErbB-3 followedby 30 unique amino acids. p75-sErbB3 is encoded by a 2.3 kb alternatec-erbB3 transcript (cDNA clone R35F) that is translated into a 515 aaprotein composed of subdomains I through III, and has a unique 41 aminoacid carboxy-terminal sequence (FIG. 1) (Lee and Maihle 1998).

A recombinant dominant-negative ErbB3 mutant with a deleted cytoplasmicdomain but which retains its transmembrane domain can inhibitfull-length ErbB2 and ErbB3 receptor phosphorylation and signaltransduction (Ram, Schelling et al. 2000). In avian tissues, expressionof a naturally occurring sEGFR/ErbB1 inhibits TGFα dependent cellulartransformation (Flickinger, Maihle et al. 1992). An aberrant solubleEGFR/sErbB1 secreted by the A431 human carcinoma cell line has beenreported to inhibit the kinase activity of full-length EGFR in aligand-independent manner (Basu, Raghunath et al. 1989). Similarly,herstatin, a naturally occurring soluble ErbB2 isoform which inhibitsErbB2 receptor phosphorylation and signaling, appears to function byblocking ErbB2 dimerization (Doherty, Bond et al. 1999). In no case dothese soluble ErbB isoforms function as antagonists of ErbB receptorsignaling through competitive, high affinity ligand binding.

Soluble ErbB3 proteins, for example p85-sErbB3 and p45-sErbB3, areunique among other naturally occurring soluble ErbB isoforms in thatthey bind specifically to heregulin with high affinity. Consequently,sErbB3 inhibits heregulin binding to cell surface ErbB receptors andheregulin-induced activation of the ErbB receptors and their downstreameffectors. Thus sErbB3, such as p85-sErbB3 and p45-sErbB3, can be usedas therapeutic reagents for heregulin-regulated malignancies such asmammary, prostate, ovary, and lung tumors. In addition, through receptorcross-talk, as well as through interactions with heterologous cellsurface receptors, these soluble receptor isoforms may also be importantfor nonheregulin-associated conditions.

SUMMARY OF THE INVENTION

Embodiments of the present invention pertain to several novel isolatedand purified nucleic acid sequences which encode soluble isoforms ofErbB3 (sErbB3). For example, embodiments of this aspect of the inventioninclude nucleic acid sequences which specifically encode a soluble formof ErbB3 whose amino acid sequence comprises the sequence of SEQ ID NO:2 or SEQ ID NO: 4. The related nucleic acid sequence embodimentscomprise SEQ ID NO: 1 and SEQ ID NO: 3, respectively.

Various embodiments of the present invention relate to an sErbB3 agentfor expression of sErbB3 or regulation of sErbB3 function or expression(either transcription or translation). Examples of an sErbB3 agentinclude without limitation a polypeptide, a nucleic acid sequence (RNAor DNA, sense or antisense), an sErbB3 antibody, an expression vector, asmall molecule, or a polypeptide agonist or antagonist.

Various embodiments of the present invention pertain to methods fortreating cancer comprising an sErbB3 agent. Other embodiments relate tosErbB3 agents as cancer therapeutics. Additional embodiments of thepresent invention relate to isoforms of sErbB3 that bind to heregulin(HRG) with high affinity and effectively block HRG binding to cellsurface receptors. For example, p85-sErbB3 binding to HRG with highaffinity substantially blocks HRG binding to cell surface receptors.Other embodiments of the present invention pertain to the use of ansErbB3 agent to regulate ErbB receptor signal transduction pathways andbiological effects on cellular proliferation, differentiation, andmetastasis. Embodiments of the present invention also pertain to therisk assessment and prevention, screening, diagnosis, prognosis,theragnosis, treatment, and evaluation of responsiveness to treatment,and monitoring of disease progression, recurrence, or metastasis ofcancer cells using sErbB3 isoforms, such as p45-sErbB3 or p85-sErbB3.

Another embodiment of the present invention pertains to an expressionvector, such as a plasmid or virus, containing an isolated cDNA encodingan sErbB3 isoform, such as p45-sErbB3 or p85-sErbB3, as well as aeukaryotic or prokaryotic cell containing the expression vector.

Embodiments of the present invention also pertain to a process forproducing the p85-sErbB3 isoform and other sErbB3 isoforms, whichcomprises the steps of ligating the isolated cDNA into an expressionvector capable of expressing the isolated cDNA in a suitable host;transforming the host with the expression vector; culturing the hostunder conditions suitable for expression of the isolated cDNA andproduction of the p85-sErbB3 protein or other sErbB3 isoforms, andisolating the protein from the host. The host cell may be a prokaryoteor a eukaryote.

Additional embodiments of the present invention relate to molecularprobes and antibody reagents specific for the unique region ofp85-sErbB3 or other sErbB3 isoform epitopes. For example, polyclonal andmonoclonal antibodies specific to p85-sErbB3 are generated using aunique C-terminal sequence of the p85-sErbB3 as an antigen. Theaffinity-purified antibody can be used to detect p85-sErbB3 usingimmunoblot, immunohistochemistry, immunoassay and other detectionmethods. One embodiment of the present invention pertains toimmunoprecipitation followed by immunoblot analysis to detect p85-sErbB3using anti-sErbB3 antibodies.

Another embodiment of the invention relates to a system and method ofdetecting p85-sErbB3 and other sErbB3 isoforms in a mammalian biologicalspecimen selected from the group consisting of fluids, such as saliva,blood, serum, plasma, urine and ascites, solid tissues, and theirderivatives.

Yet another embodiment of the invention relates to a vector for genetherapy, comprising a nucleic acid molecule having i) a transcriptionregulatory sequence; and ii) a second sequence coding for p85-sErbB3 oranother sErbB3 isoform under transcriptional control of thetranscription regulatory sequence; and iii) a delivery vehicle fordelivering the nucleic acid molecule.

Those and other details, objects, and advantages of the presentinvention will become better understood or apparent from the followingdescription and drawings showing embodiments thereof.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings illustrate examples of embodiments of theinvention. In such drawings:

FIG. 1. is a diagram of soluble ErbB3 (sErbB3) proteins. ErbB3 iscomposed of a 19 amino acid (aa) signal peptide sequence that is cleaved(gray box), an extracellular ligand-binding domain (aa 1-620), atransmembrane domain (aa 621-646; indicated as TM), and an intracellulardomain (aa 647-1323). The extracellular domain of the receptor can befurther divided into four subdomains (I-IV), as noted in the text. Thealternate c-erbB3 transcripts arise from read-through of an intron andthe use of alternative polyadenylation signals. p45-sErbB3 contains theamino-terminal 310 amino acids of ErbB3 and two unique carboxy-terminalamino acid residues. p50-sErbB3 contains the amino-terminal 351 aminoacids of ErbB3 and 30 unique carboxy-terminal amino acid residues.p75-sErbB3 contains the amino-terminal 474 amino acids of ErbB3 and 41unique carboxy-terminal amino acid residues. p85-sErbB3 contains theamino-terminal 519 amino acids of ErbB3 and 24 unique carboxy-terminalamino acid residues. The carboxy-terminal unique sequences are denotedas black boxes.

FIG. 2. demonstrates that p45-sErbB3 and p85-sErbB3 in conditioned mediacan block HRG-induced activation of ErbB3. (A) p45-sErbB3 and p85-sErbB3in the concentrated conditioned media were detected by Western blottingusing an anti-ErbB3 antibody recognizing the extracellular region ofErbB3. Increasing volumes (5, 10, 20 μl; left to right) of theconcentrated conditioned media (×15) were loaded on an SDS-PAGE gel. (B)and (C) The Ba/F3 (ErbB2+ErbB3) cells were stimulated with HRGα (panelB) and HRGβ (panel C) with or without the concentrated conditioned mediafor 10 min at room temperature prior to lysis. ErbB3 wasimmunoprecipitated with an anti-ErbB3 antibody from equal amounts oftotal protein, subjected to SDS-PAGE, and analyzed by Western blottingusing an anti-phosphotyrosine antibody (αPY). Filters were stripped andreprobed with anti-ErbB3 antibody recognizing the intracellular regionof ErbB3.

FIG. 3. demonstrates p85-sErbB3 binds to HRG. (A) HRGβ was crosslinkedto p85-sErbB3 (25 nM) with BS³ after incubating in the presence of 50 nM¹²⁵I-HRGβ without or with increasing concentrations (0.16, 0.32, 0.64,1.25 μM) of unlabeled HRGβ. Insulin (1.25 μM) was used as a negativecontrol. The arrowhead indicates a 90 kDa complex of ¹²⁵I-HRGβ andp85-sErbB3. (B) and (C) Binding analysis of ¹²⁵I-HRG to p85-sErbB3 andErbB3-IgG fusion protein. Binding assays were performed in a 96-wellplate format as described below in more detail in the Examples. Bindingresults were analyzed by using Scatchard method and by plotting thedisplacement of ¹²⁵I-HRGβ₁₇₇₋₂₄₄ binding by unlabeled HRGβ₁₇₇₋₂₄₄(Inset).

FIG. 4. is a graph showing inhibition of HRGβ binding by p85-sErbB3 andby 2C4, a monoclonal antibody specific for ErbB2. T47D cells wereincubated with the indicated concentrations of p85-sErbB3 and 2C4 atroom temperature for 30 min. ¹²⁵I-HRGβ₁₇₇₋₂₄₄ (0.1 nM) was then addedand binding reactions were performed as described below in more detailin the Examples. ¹²⁵I-HRGβ₁₇₇₋₂₄₄ bound to the cell surface was measuredusing a gamma counter.

FIG. 5. demonstrates p85-sErbB3 blocks HRG-induced activation of ErbB2and ErbB3 in the Ba/F3 (ErbB2+ErbB3) cells. Cells were untreated orstimulated with HRGβ₁₋₂₄₁ alone or HRGβ₁₋₂₄₁ plus purified p85-sErbB3for 10 min at room temperature. Receptor phosphorylation levels andErbB2 and ErbB3 receptor levels were determined by anti-ErbB2 (A) andanti-ErbB3 (B) immunoprecipitation followed by Western blotting asdescribed in FIG. 2.

FIG. 6. demonstrates p85-sErbB3 blocks HRG-induced activation of ErbBproteins and their downstream activators MAPK, PI3K (p85), and Akt. (A)p85-sErbB3 blocks HRG-induced activation of ErbB2, ErbB3, and ErbB4 inT47D and MCF7 breast carcinoma cells. Serum-starved cells werestimulated with no HRGβ, HRGβ alone, or 6 nM HRGβ plus 36 nM p85-sErbB3for 10 min at room temperature. Receptor phosphorylation levels andErbB2, ErbB3, and ErbB4 receptor levels were determined byimmunoprecipitation followed by Western blotting. (B) p85-sErbB3inhibits HRG-induced association of PI3K (p85) with ErbB3 and activationof MAPK and Akt in T47D cells. Cells were treated with 1 nM HRGβ and 10nM p85-sErbB3 for 10 min or 30 min and analyzed for activation of ErbB3.Association of PI3K (p85) with ErbB3 was analyzed by immunoprecipitationof cell lysates using an anti-ErbB3 antibody followed by Westernblotting of anti-PI3K (p85) antibody. Activation of MAPK and Akt wasexamined by Western blotting of cell lysates using antibodies specificto phospho-MAPK and phospho-Akt.

FIG. 7. demonstrates p85-sErbB3 inhibits cell growth stimulation by HRG.MCF7 cells were trypsinized, washed, and plated at a density of 5,000(squares) or 8,000 cells/well (triangles) in 96-well plates withincreasing concentrations of HRGβ in serum-free medium and growth wasmeasured after 3 days (inset). MCF7 cells were trypsinized, washed,incubated with p85-sErbB3 for 30 min, and plated with or without 0.4 nMHRGβ in serum-free medium. At 40 nM (a 100-fold molar excess to HRGβ) inthe presence of HRGβ, p85-sErbB3 inhibited cell growth by 75% and 90%,at densities of 5,000 and 8,000 cells/well, respectively, whereas thesame concentration of p85-sErbB3 did not affect cell growth in theabsence of HRGβ. The data presented are the mean±standard deviation ofsix replicates. This experiment was repeated three times and the resultsshown represent all three trials.

FIG. 8 is an immunoblot demonstrating specificity of the p85-sErbB3antibody.

DESCRIPTION OF THE INVENTION

As used herein, the term “soluble” ErbB3 (sErbB3) means that thepolypeptide is found in a form that is not anchored to the membrane of acell via a typical transmembrane domain, i.e., a portion of the sErbB3is not found physically embedded in the lipid bilayer which comprisesthe cell membrane.

As used herein, the term “biological activity” is defined to mean apolypeptide or a variant or fragment thereof, which has at least about10%, preferably at least about 50%, and more preferably at least about90% of the biological activity of the related polypeptide, such as forexample the proteins having the amino acid sequence of SEQ ID NO:2 orSEQ ID NO: 4. The biological activity of a polypeptide of the inventioncan be measured by methods well known in the art including, but notlimited to, the ability to bind heregulins or inhibit ErbB receptormediated signaling.

As used herein, the term “protein” is a polypeptide, and the term“polypeptide” comprises at least two amino acids with no predefinedlimitation in length. sErbB3 polypeptides may be the complete sequence,for example SEQ ID NO:2 or SEQ ID NO:4, or may be correspondingfragments or variants thereof, such as the unique carboxy terminalregion.

The terms “recombinant nucleic acid” or “preselected nucleic acid,”“recombinant DNA sequence or segment” or “preselected DNA sequence orsegment” refer to a nucleic acid that has been derived or isolated fromany appropriate tissue source and that may be subsequently chemicallyaltered, typically in vitro, so that its sequence is not naturallyoccurring, or corresponds to naturally occurring sequences that are notpositioned as they would be positioned in a genome which has not beentransformed with exogenous DNA. An example of preselected DNA “derived”from a source would be a DNA sequence that is identified as a usefulfragment within a given organism and which is then chemicallysynthesized in essentially pure form. An example of such DNA “isolated”from a source would be a useful DNA sequence that is excised or removedfrom the source by chemical means, e.g., by the use of restrictionendonucleases, so that it can be further manipulated, e.g., amplifiedfor use in the invention by the methodology of genetic engineering.

“Regulatory sequences” is defined to mean RNA or DNA sequences necessaryfor the expression, post-transcriptional modification, translation, andpost-translational modification of an operably linked coding sequence ina particular host organism. The control sequences that are suitable forprokaryotic cells, for example, include a promoter, and optionally anoperator sequence, and a ribosome binding site. Eukaryotic cells areknown to utilize promoters, stop sequences, enhancers, splicing, andpolyadenylation signal sequences, as well as glycosylation and secretorysignal sequences.

As used herein, the term “cell line” or “host cell” is intended to referto well-characterized homogenous, biologically pure populations ofcells. These cells may be eukaryotic cells that are neoplastic or whichhave been “immortalized” in vitro by methods known in the art, as wellas primary cells, or prokaryotic cells. The cell line or host cell ispreferably of mammalian origin, but cell lines or host cells ofnon-mammalian origin may be employed, including avian, plant, insect,yeast, fungal or bacterial sources. Generally, the preselected DNAsequence is related to a DNA sequence that is resident in the genome ofthe host cell but is not expressed, or not highly expressed, or,alternatively, over-expressed.

The terms “transfected” or “transformed” are used herein to include anyhost cell or cell line, the genome of which has been altered oraugmented by the presence of at least one preselected DNA sequence,which DNA is also referred to in the art of genetic engineering as“heterologous DNA,” “recombinant DNA,” “exogenous DNA,” “geneticallyengineered DNA,” “non-native DNA,” or “foreign DNA,” wherein the DNA wasisolated and introduced into the genome of the host cell or cell line bythe process of genetic engineering. The host cells of the presentinvention are typically produced by transfection with a DNA sequence ina plasmid expression vector, a viral expression vector, or as anisolated linear DNA sequence. Preferably, the transfected DNA is achromosomally integrated recombinant DNA sequence which comprises a DNAencoding an sErbB3 isoform, for which the host cell may or may notexpress significant levels of autologous or “native” sErbB3.

A recombinant, functionally dominant-negative ErbB3 mutant with adeleted cytoplasmic domain, but which retains its transmembrane domain,can inhibit full-length ErbB2 and ErbB3 receptor activation (Ram, T. G.,et al., 2000). In avian tissues, expression of a naturally occurringsEGFR/sErbB1 isoform inhibits TGFα dependent transformation. An aberrantsoluble EGFR secreted by the A431 human carcinoma cell line also hasbeen shown to inhibit the kinase activity of full-length EGFR. Thesesoluble EGFR/ErbB1 receptors do not function as antagonists through highaffinity ligand-binding. Herstatin, a naturally occurring soluble ErbB2isoform inhibits ErbB2 receptor activation by blocking ErbB2dimerization. In contrast, sErbB3, for example p85-sErbB3 andp45-sErbB3, inhibits HRG-induced stimulation of ErbB2, ErbB3, and ErbB4,at least in part, by neutralizing ligand activity through competitivebinding.

The physiological role of p85-sErbB3 in normal tissues also has not beenunderstood to date. As discussed in greater detail below, although amuch higher concentration (100-fold) was required to inhibit cellgrowth, a 10-fold molar excess of p85-sErbB3 was sufficient to inhibitErbB receptor phosphorylation. At this ratio of sErbB3 to full-lengthErbB receptor, cell growth is stimulated by a small fraction ofactivated ErbB receptors that are still activated and sufficient forgrowth stimulation. It is known that the 2.1 kb transcript encodingp85-sErbB3 is expressed at low levels compared to the full-lengthc-erbB3 transcript in all cell lines and tissues examined to date.Research shows that local concentrations of autocrine growth factorssuch as EGF are exquisitely regulated and do not travel far from thecell surface from which they are released to exert their biologicaleffects. In this context, tightly regulated localized concentrations ofp85-sErbB3 have important consequences on cellular activities, such asHRG-mediated cell growth. These effects on cell growth are even moredramatic in cancer cells where cell polarity is typically lost,resulting in deregulation of normal spatial and temporal control ofgrowth factor growth factor receptor interactions because normal spatialcellular barriers are disrupted.

The present invention provides several novel isolated and purifiednucleic acid sequences which encode isoforms of sErbB3 and nucleic acidsequences encoding engineered variants of these proteins. For example,disclosed herein are nucleic acids which specifically encode isoforms ofsErbB3 whose amino acid sequences comprise the sequences of SEQ ID NO: 2and SEQ ID NO: 4. Embodiments of the present invention relate to the useof sErbB3 as a unique HRG inhibitor because it can block HRG binding tocell surface ErbB receptors via binding to HRG with high affinity,thereby inhibiting HRG-induced stimulation of ErbB2, ErbB3, and ErbB4.This inhibition is sufficient to effectively block HRG-stimulated cellgrowth. These novel sErbB3 isoforms, therefore, are potent modulators ofHRG regulated cell functions, such as proliferation, migration, anddifferentiation in normal mammalian and human tissues. Embodiments ofthe present invention also relate to regulation, either upregulation ordownregulation, of heregulin activity using an sErbB3 agent.

Various embodiments of the invention relate to an sErbB3 agent forexpression of sErbB3 or regulation, either upregulation ordownregulation, of sErbB3 activity or expression (either transcriptionor translation). Examples of an sErbB3 agent include without limitationa polypeptide, a nucleic acid sequence (RNA or DNA, sense or antisense),an sErbB3 antibody, an expression vector, a small molecule, or apolypeptide agonist or antagonist.

Soluble ErbB3 also binds to ErbB receptors and regulates ErbB receptorsignaling activities through downstream effectors such as MAPK, Akt, andPI3K. Additional embodiments of the present invention relate toregulation, either upregulation or downregulation, of ErbB receptoractivities using an sErbB3 agent.

As further described below in the examples, sErbB3 is expressed in mostepithelial tissues of many organs, including esophagus, liver, colon,stomach, thyroid, head and neck, kidney, bladder, pancreas, lung, skin,breast, ovary, uterus (cervix and endometrium), prostate gland, brain,intestine, or testis. Various embodiments of the present invention alsorelate to methods for treating cancer, such as carcinomas and gliomas,and cancer therapeutics comprising sErbB3 agents which regulate sErbB3expression and/or function.

Nucleic Acid and Amino Acid Sequences.

The polypeptides in embodiments of the present invention may be insubstantially pure form, isolated as a recombinant form, and may befused to other moieties. Additional N-terminal or C-terminal sequencesmay be added to the polypeptides for various reasons, for example toimprove expression or regulation of expression in particular expressionsystems, to provide protection against proteolytic cleavage, or to aidin identification as fusion proteins or purification such as affinitychromatography using fusion proteins. Techniques for providing suchadditional sequences are well known in the art. Furthermore polypeptideswith additional N-terminal or C-terminal sequences may simply resultfrom the technique used to obtain the polypeptide without providing anyadvantageous characteristics and are also within the scope of thepresent invention. Whatever sequence is added, the resultant polypeptidepreferably exhibits the biological activity of the cognate sErbB3, forexample a polypeptide having the amino acid sequence SEQ ID NO: 2 or SEQID NO: 4.

The polypeptides of the present invention include post-translationalmodifications, for example and without limitation, phosphorylation,glycosylation and farnesylation.

Furthermore, alterations, either conservative or non-conservative, canoccur in the amino acid sequence of a polypeptide, which likely do notaffect the function. Such alterations include amino acid deletions,insertions, and substitutions. Such alterations can result fromalternative splicing and/or the presence of multiple translation startsites and/or stop sites. Polymorphisms may also arise as a result of theinfidelity which is inherent in the translation process. Conservativesubstitutions are preferred. As such, the amino acids glycine, alanine,valine, leucine and isoleucine are often substituted for one another. Ofthese possible substitutions, it is preferred that glycine and alanineare used to substitute for one another because they have relativelyshort aliphatic side chains and that valine, leucine and isoleucine areused to substitute for one another because they have larger hydrophobicaliphatic side chains. Other amino acids that are often substituted forone another include: phenylalanine, tyrosine and tryptophan (amino acidshaving aromatic side chains); lysine, arginine and histidine (aminoacids having basic side chains); aspartate and glutamate (amino acidshaving acidic side chains); asparagine and glutamine (amino acids havingamide side chains); and cysteine and methionine (amino acids havingsulphur-containing side chains). Aspartic acid and glutamic acid cansubstitute for phosphoserine and phosphothreonine, respectively (aminoacids with acidic side chains). Amino acid substitutions or insertionscan be made using naturally occurring or non-naturally occurring aminoacids; however, L-amino acids are preferred in one embodiment.

Whatever amino acid changes are made, whether by means of substitution,modification, insertion or deletion, polypeptides embodied withinembodiments of the present invention have at least 50% sequence identitywith the related sErbB3 isoform, for example isoforms comprising SEQ IDNO: 2 or SEQ ID NO: 4, and in various embodiments the degree of sequenceidentity is at least 75%. In various embodiments, sequence identities ofat least 80%, 85%, 90%, 95%, 98% or 99% are used.

Amino acid sequence variants of an sErbB3 or fragments thereof can beprepared by a number of techniques, such as random mutagenesis of DNAwhich encodes an sErbB3 or a region thereof. Useful methods also includePCR mutagenesis, cassette mutagenesis, and saturation mutagenesis. Alibrary of random amino acid sequence variants can also be generated bythe synthesis of a set of degenerate oligonucleotide sequences.Non-random or directed mutagenesis techniques can be used to providespecific sequences or mutations in specific regions. Alanine scanningmutagenesis is a useful method for identification of certain residues orregions of the desired protein that are preferred locations or domainsfor mutagenesis, see for example Cunningham and Wells (Science244:1081-1085, 1989). Oligonucleotide-mediated mutagenesis is a usefulmethod for preparing substitution, deletion, and insertion variants ofDNA, see, e.g., Adelman et al., (DNA 2:183, 1983). Additionally, inanother example, combinatorial mutagenesis is used to generate variants.

Embodiments of the present invention further relate to variants of thenucleic acid sequences, for example nucleic acid sequences SEQ ID NO:1and SEQ ID NO: 3, which encode proteins, analogs or derivatives of ansErbB3, for example p85-sErbB3 and p45-sErbB3. Such nucleic acidvariants are produced by nucleotide substitutions, deletions, oradditions and may involve one or more nucleotides.

In examples, the nucleic acid sequence is a genomic sequence or a cDNAsequence. The nucleotide sequence includes, for example: an sErbB3coding region; a promoter sequence, such as a promoter sequence from ansErbB3 gene or from another gene; an enhancer sequence; untranslatedregulatory sequences either 5′ or 3′ from an sErbB3 gene or from anothergene; a polyadenylation site; and an insulator sequence. The nucleotidesof embodiments of the present invention can be modified at the basemoiety, sugar moiety or phosphate backbone to improve the stability,hybridization, or solubility of the molecules. For instance, thedeoxyribose phosphate backbone of the polynucleotide molecules ismodified to generate peptide polynucleotides (see, for example Hyrup etal, Bioorganic & Medicinal Chemistry, 4:523, 1996). As used herein, theterms “peptide polynucleotides” or “PNAs” refer to polynucleotidemimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. In examples, PNA oligomers are synthesized usingstandard solid phase peptide synthesis protocols. PNAs are used intherapeutic and diagnostic applications. For example, PNAs are used asantisense agents for sequence-specific modulation of gene expression.

In other examples, the unique sErbB3 sequences are also used as a targetfor selective inhibition of expression (stability, transcription, ortranslation) using siRNA, RNAi, short hairpin RNA, microRNAs, ribozyme,and triple helix methodologies, as well as antisense sequences,including antisense oligonucleotides. Useful fragments of the sErbB3nucleic acid sequences include antisense or sense oligonucleotidescomprising a single-stranded nucleic acid sequence (either RNA or DNA)capable of binding to target sErbB3 mRNA or sErbB3 DNA sequences.Antisense or sense oligonucleotides comprise a fragment of the codingregion of an sErbB3, for example to a unique region such as theC-terminus. Such a fragment generally comprises at least about 5nucleotides, and typically 14 to 30 nucleotides. The ability to derivean antisense or a sense oligonucleotide, based upon a cDNA sequenceencoding a given protein is well known in the art and is described in,for example, Stein and Cohen (Cancer Res. 48:2659, 1988), van der Krolet al. (Bio Techniques 6:958, 1988), Izant J. G. and Weintraub H.,(Cell, 36: 100.7-1015, 1984) and Rosenberg et al. (Nature, 313:703-706,1985).

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block transcriptionor translation of the target sequence by one of several means, includingenhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. Thus, the antisenseoligonucleotides are used for example to block expression of sErbB3proteins. Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones or othersugar linkages, and wherein such sugar linkages are resistant toendogenous nucleases. Such oligonucleotides with resistant sugarlinkages are stable in vivo, i.e., capable of resisting enzymaticdegradation, but retain sequence specificity to be able to bind totarget nucleotide sequences.

Other examples of sense or antisense oligonucleotides includeoligonucleotides which are covalently linked to other organic moieties,such as for example organic moieties that increase affinity of theoligonucleotide for a target nucleic acid sequence, such aspoly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

In other examples, sense or antisense oligonucleotides are introducedinto a cell containing the target nucleotide sequence by formation of aconjugate with a ligand binding molecule, for example cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

These sense and antisense nucleic acid sequences have utility astherapeutic agents, in methods of treating cancer or other diseases andmedical conditions, and in methods of regulating sErbB3 expression.

The sequences embodied herein relate specifically to sErbB3 isoforms,for example SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8. Variousembodiments relate to the unique C-terminal region of the sErbB3isoforms, for example variants and fragments specific to the uniqueregion.

Expression Vectors and Methods of Delivery

Other embodiments of the present invention comprise an expression vectorcontaining a nucleic acid sequence that expresses an sErbB3 polypeptide,for example a polypeptide having an amino acid sequence comprising SEQID NO:2 or SEQ ID NO:4, in a suitable host. In an example, the nucleicacid sequence has a promoter operably linked to the polypeptide codingregion, the promoter being inducible or constitutive and, optionally,cell-type or tissue-specific. In an example, the promoter may also be aheterologous promoter. The vector may be, for example, a plasmid, asingle or double-stranded phage vector, or a single or double-strandedviral RNA or DNA molecule. An example of an inducible vector is a vectorinduced for expression by environmental factors that are easy tomanipulate, such as temperature and nutrient additives. Examples ofviral vectors include viruses such as baculoviruses, papova viruses suchas SV40, vaccinia viruses, adenoviruses including adeno-associatedviruses, fowl pox viruses, lentiviruses, parvoviruses, herpes simplexviruses, pseudorabies viruses, and retroviruses, as well as vectorsderived from combinations thereof, such as those derived from plasmidand bacteriophage genetic elements, such as cosmids and phagemids.

Embodiments of the present invention also include therapeutic expressionof genetic material, including gene therapy expression of an sErbB3isoform, variant, or fragment. Gene therapy is either by in vivo genetherapy, which is direct delivery of the nucleic acid or nucleicacid-carrying vector into a patient, or ex vivo gene therapy, which isindirect delivery to the patient via transplanted cells that were firsttransformed with the nucleic acid sequences or nucleic acid-carryingvector in vitro. In examples, viral vectors such as the examples listedherein can be used for in vivo and ex vivo gene therapy.

Plasmid DNA can be delivered with the help of, for example and withoutlimitation: cationic liposomes such as lipofectin, or derivatized (e.g.antibody conjugated) polylysine conjugates, nanoparticles, gramacidin S,artificial viral envelopes or other such intracellular carriers, as wellas direct injection of the gene construct or CaPO.sub.4 precipitationcarried out in vitro.

In other examples, a subject polynucleotide is administered using anon-viral delivery vehicle. “Non-viral delivery vehicle” (also referredto herein as “non-viral vector”) as used herein is meant to includechemical formulations containing naked or condensed polynucleotides(e.g., a formulation of polynucleotides and cationic compounds, forexample dextran sulfate), and naked or condensed polynucleotides mixedwith an adjuvant such as a viral particle (i.e., the polynucleotide ofinterest is not contained within the viral particle, but thetransforming formulation is composed of both naked polynucleotides andviral particles, see, for example Curiel et al. 1992 Am. J. Respir. CellMol. Biol. 6:247-52. Thus “non-viral delivery vehicle” includes vectorscomposed of polynucleotides plus viral particles where the viralparticles do not contain the polynucleotide of interest. “Non-viraldelivery vehicles” include bacterial plasmids, viral genomes or portionsthereof, wherein the polynucleotide to be delivered is not encapsidatedor contained within a viral particle, and constructs comprising portionsof viral genomes and portions of bacterial plasmids and/orbacteriophages. The term also encompasses natural and synthetic polymersand co-polymers. The term further encompasses lipid-based vehicles.Lipid-based vehicles include cationic liposomes such as disclosed forexample by Felgner et al (U.S. Pat. Nos. 5,264,618 and 5,459,127; PNAS84:7413-7417, 1987; Annals N.Y. Acad. Sci. 772:126-139, 1995), DDAB,DOPC, and phospholipids such as phophatidylcholine. In other examples,lipid based vehicles consist of neutral or negatively chargedphospholipids or mixtures thereof including artificial viral envelopesas disclosed for example by Schreier et al. (U.S. Pat. Nos. 5,252,348and 5,766,625).

Non-viral delivery vehicles include polymer-based carriers, includingnatural and synthetic polymers and co-polymers. Preferably, the polymersare biodegradable or are readily eliminated from the subject. Naturallyoccurring polymers include polypeptides and polysaccharides. Syntheticpolymers include, but are not limited to, polylysines andpolyethyleneimines (PEI; see for example Boussif et al., PNAS92:7297-7301, 1995) which molecules can also serve as condensing agents.These carriers are dissolved, dispersed or suspended in a dispersionliquid such as water, ethanol, saline solutions and mixtures thereof. Awide variety of synthetic polymers are known in the art.

In examples, naked DNA or RNA molecules are used where they are in aform which is resistant to degradation, such as by modification of theends, by the formation of circular molecules, or by the use of alternatebonds including phosphothionate and thiophosphoryl modified bonds. Inother examples, the delivery of nucleic acids is facilitated bytransport where the nucleic acid molecules are conjugated to polylysineor transferrin.

Antisense or sense oligonucleotides are introduced into a cellcontaining the target nucleic acid sequence by any genetic materialtransfer method, including, for example, CaPO.sub.4-mediated DNAtransfection, lipid mediated transfection, electroporation, or by usinggene transfer vectors or methods described above. In another example, anantisense or sense oligonucleotide is inserted into a suitable viralvector, such as those described previously. A cell containing the targetnucleic acid sequence is contacted with the recombinant viral vector,either in vivo or ex vivo.

Detection/Diagnosis

Other embodiments of the present invention relate to methods fordetecting an sErbB3 and assessing the risk of developing a preneoplasticlesion or cancer, screening for a cancer, or diagnosing a cancer.Additional embodiments of the present invention relate to assaying abiological sample for an sErbB3 to evaluate prognosis, theragnosis,responsiveness to a treatment of cancer, prophylactic selection of acancer prevention regimen, early detection of a cancer, or cancerprogression, recurrence, or metastasis. One embodiment relates to amethod of assaying for an sErbB3 and/or diagnosing a cancer in asubject, for example a human or other mammal, which comprises the stepof detecting and/or quantifying a concentration of a polypeptide ornucleic acid sequence of the invention in a biological sample obtainedfrom said subject. Examples of biological samples include fluids, suchas saliva, blood, serum, plasma, urine and ascites, solid tissues, andtheir derivatives. In an example, antibodies which recognize apolypeptide of the invention are used to detect the amount of thepolypeptide in a biological sample such as serum.

In one embodiment, binding of an sErbB3 antibody in tissue sections canbe used to detect aberrant polypeptide localization or an aberrantconcentration of polypeptide. In another embodiment, an antibody to apolypeptide of the invention can be used to assay a subject sample, forexample tissue or serum, for the concentration of the polypeptide wherean aberrant amount of polypeptide is indicative of a risk of developinga preneoplastic lesion or cancer, prognosis, theragnosis, responsivenessto treatment, prophylactic selection of a cancer prevention regimen,early detection of a cancer, or cancer progression, recurrence, ormetastasis. As used herein, an “aberrant amount” means an amount that isincreased or decreased compared with the amount in a subject free fromcancer or an established reference level.

Examples of suitable immunoassays for detecting or assaying sErbB3include, without limitation, competitive and non-competitive assaysystems using techniques such as western blots, radioimmunoassays, ELISA(enzyme linked immunosorbent assay), acridinium linked immunosorbentassays (ALISA), “sandwich” immunoassays, immunohistochemical assays,immunofluorescent detection assays, immunoprecipitation assays,precipitin reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays and protein Aimmunoassays. Immunoassays to detect and quantitate an sErbB3, forexample p85-sErbB3, have been developed for use on formalin-fixed,paraffin-embedded tissue and tumor samples and for use on frozen-sectiontissue and tumor samples. In addition, antibodies can be used toselectively quantitate an sErbB3 polypeptide in other tissues, includingfor example saliva, blood, serum, plasma, and urine, using enzyme-linkedimmunosorbent assays, acridinium-linked immunosorbent assays, andradioimmunoassay. Such assays may be combined with the quantitiveassessment of other biomarkers on a similar platform (e.g., multiplexassays) to increase the biological or clinical information obtained.

Quantitation of the expression of the specific mRNA encoding thepolypeptide can be performed using methods such as RNA in situhybridization (RNA ISH), as well as other complementary RNAmethodologies, such as RNAse protection assays. Genetic aberrations inthe relative copy number of the sequence can be performed by any genomicDNA detection method, for example FISH.

Antibodies

Soluble ErbB3 polypeptides embodied within the present invention can be“antigenic” and/or “immunogenic”. Generally, “antigenic” means that thepolypeptide is capable of being used to generate antibodies or indeed iscapable of inducing an antibody response in a subject. “Immunogenic”means that the polypeptide is capable of eliciting an immune response ina subject. For example, the polypeptide could not only generate anantibody or anti-idiotypic antibody response but, in addition,non-antibody based immune responses, and also could be used to produce atherapeutic vaccine. In an example, the unique C-terminal region of ansErbB3 is a target of the vaccine.

Further embodiments relate to antibodies, which specifically bind ansErbB3 isoform, generated using an immunogen derived from the sErbB3isoform, such as from an isoform having SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, or SEQ ID NO: 8. In an example, an antibody specific to thesErbB3 is generated using the unique C-terminal region (describedherein) of the specific sErbB3, such as amino acids 540-562 of SEQ IDNO: 2, a 23 aa sequence (Ser Lys Gly Ser Gln Ser Arg Met Gly Gly Gly GlyAla Leu Gln Trp Asn Cys Ser Gly Gly Ile Gln) from the unique C-terminalsequence of p85-sErbB3. Such antibodies include, but are not limited topolyclonal, monoclonal, bispecific, humanized or chimeric antibodies,single chain antibodies, Fab fragments and)F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, affibodies, and epitope-binding fragments of any of theabove. As used herein, “antibody” refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that specifically bindsan antigen. The immunoglobulin molecules can be of any class, e.g., IgG,IgE, IgM, IgD and IgA, or subclass of immunoglobulin molecule. Asdescribed above, such antibodies are used for detection andquantification as well as in methods of treating cancer, methods ofregulating sErbB3 isoforms, and cancer therapeutics.

In the production of antibodies, screening for the desired antibody isaccomplished by techniques known in the art, e.g., ELISA (enzyme-linkedimmunosorbent assay). In one example, to select antibodies whichrecognize a specific domain of a polypeptide of the invention, generatedhybridomas are assayed for reactivity toward a product which binds to apolypeptide fragment of the sErbB3 isoform.

Polyclonal antibodies directed towards a sErb3 polypeptide are generatedby stimulating their production in a suitable animal host (e.g. achicken, mouse, rat, guinea pig, rabbit, sheep, goat or monkey) when apolypeptide of embodiments of the present invention is injected into theanimal. If necessary, an adjuvant may be administered together with thepolypeptide of the invention. The antibodies are then purified by virtueof high affinity binding to the associated polypeptide of the invention.

Monoclonal antibodies (mAbs) directed toward an sErbB3 polypeptide maybe generated by any technique known to those skilled in the art toprovide for the production of antibody molecules by continuous celllines in culture. Some examples for producing mAbs include the hybridomatechnique (Kohler and Milstein, 1975, Nature 256:495-497), the triomatechnique, the human B-cell hybridoma technique (Kozbor et al., 1983,Immunology Today 4:72), the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al., 1985, Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96), and germ-free animals(PCT/US90/02545). The hybridoma producing the mAbs of the invention maybe cultivated in vitro or in vivo. The mAbs to sErbB3 include but arenot limited to human mAbs and chimeric mAbs (e.g., human-mousechimeras). A chimeric antibody is a molecule in which different portionsare derived from different animal species, such as those having a humanimmunoglobulin constant region and a variable region derived from amurine mAb. See, for example, U.S. Pat. No. 4,816,567; and U.S. Pat. No.4,816,397. Humanized antibodies are antibody molecules from non-humanspecies having one or more complementarity determining regions (CDRs)from the non-human species and a framework region from a humanimmunoglobulin molecule. See, for example, U.S. Pat. No. 5,585,089.

Chimeric and humanized mAbs can be produced by recombinant DNAtechniques known in the art, for example and without limitation usingmethods described in WO 87/02671; EP 184,187; EP 171,496; EP 173,494; WO86/01533; U.S. Pat. No. 4,816,567; EP 125,023; Better et al., 1988,Science 240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al.,1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987,Canc. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; and Shawet al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985,Science 229:1202-1207; Oi et al., 1986, Bio/Techniques 4:214; U.S. Pat.No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al.(1988) Science 239:1534; and Beidler et al., 1988, J. Immunol.141:4053-4060; Neuberger et al., 1984, Nature 312:604-608; Takeda etal., 1985, Nature 314:452-454).

For therapeutics and methods of treating human patients, completelyhuman sErbB3 antibodies are desirable. Such antibodies can be generated,for example, using transgenic mice which are incapable of expressingendogenous immunoglobulin heavy and light chain genes, but which canexpress human heavy and light chain genes. The transgenic mice areimmunized by methods known to those skilled in the art with a selectedantigen, e.g., all or a portion of an sErbB3 specific polypeptide, forexample SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or aunique C-terminal sequence, such as the unique 24 aa C-terminal sequenceof p85-sErbB3 (for example, amino acids 540-562 of SEQ ID NO: 2: Ser LysGly Ser Gln Ser Arg Met Gly Gly Gly Gly Ala Leu Gln Trp Asn Cys Ser GlyGly Ile Gln). Then, mAbs directed against the antigen can be obtainedusing conventional hybridoma technology where the human immunoglobulintransgenes harbored by the transgenic mice rearrange during B celldifferentiation, and subsequently undergo class switching and somaticselection. By using such a technique, therapeutically useful IgG, IgA,IgM, IgD and IgE antibodies can be produced. For references andprotocols for producing human antibodies, see, for examples Lonberg andHuszar (1995, Int. Rev. Immunol. 13:65-93); U.S. Pat. No. 5,625,126;U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,569,825; U.S. Pat. No.5,661,016; and U.S. Pat. No. 5,545,806.

Furthermore, completely human antibodies which recognize a selectedepitope can be produced, for example, by the “guided selection”technique in which a selected non-human mAb, e.g., a mouse antibody, isused to guide the selection of a completely human antibody recognizingthe same epitope such as disclosed in jespers et al. (1994)Biotechnology 12:899-903.

The sErbB3 antibodies embodied herein also can be generated usingvarious phage display methods known in the art whereby functionalantibody domains are displayed on the surface of phage particles whichcarry the polynucleotide sequences encoding the functional antibodydomains. After phage selection, which is performed for example by usinglabeled antigen or antigen bound or captured to a solid surface or bead,the antibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria. Techniques known in the art to recombinantly produce Fab, Fab′and F(ab′)2 fragments can also be employed.

Single-chain Fvs and antibodies which bind an sErbB3 also can beproduced by methods known in the art, such as for example thosedisclosed in U.S. Pat. No. 4,946,778 and U.S. Pat. No. 5,258,498; Hustonet al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988).

Further, bispecific antibodies which bind to an sErbB3 can be made bymethods known in the art and are embodied herein. For example,bispecific antibodies comprise a hybrid immunoglobulin heavy chain witha first binding specificity in one arm, and a hybrid immunoglobulinheavy chain-light chain pair with a second binding specificity in theother arm. See, for example WO 94/04690 and Suresh et al., Methods inEnzymology, 1986, 121:210.

Embodiments of the present invention include functionally activefragments, derivatives or analogs of the anti-polypeptide immunoglobulinmolecules. “Functionally active” means that the fragment, derivative oranalogue is able to elicit anti-anti-idiotype antibodies (i.e., tertiaryantibodies) that recognize the same antigen that is recognized by theantibody from which the fragment, derivative or analogue is derived. Inan example, the antigenicity of the idiotype of the immunoglobulinmolecule is enhanced by deletion of framework and CDR sequences that areC-terminal to the CDR sequence that specifically recognizes the antigen.To determine which CDR sequences bind the antigen, synthetic peptidescontaining the CDR sequences are used in binding assays with the antigenby any binding assay method known in the art.

Embodiments of the present invention include antibody fragments such as,but not limited to, F(ab′)2 fragments and Fab fragments. In examples,antibody fragments which recognize specific epitopes are generated byknown techniques to those skilled in the art. F(ab′)2 fragments consistof the variable region, the light chain constant region and the CH1domain of the heavy chain and are generated by pepsin digestion of theantibody molecule. Fab fragments are generated by reducing thedisulphide bridges of the F(ab′).sub.2 fragments. Further, any othermolecule with the same specificity as the antibodies and antibodyfragments of embodiments of the present invention are embodied herein.

Embodiments of the present invention also relate to heavy chain andlight chain dimers of the antibodies of the invention, or any minimalfragment thereof such as Fvs or single chain antibodies (SCAs). See forexample U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:42342; Hustonet al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al.,1989, Nature 334:544-54. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide. Techniques for theassembly of functional Fv fragments in E. coli may be used such as themethod disclosed in Skerra et al., (1988, Science 242:1038-1041).

Additional embodiments of the present invention provide for fusionpolypeptides of the immunoglobulins of embodiments of the invention, orfunctionally active fragments thereof, for example in which theimmunoglobulin is fused via a covalent bond (e.g., a peptide bond), ateither the N-terminus or the C-terminus to an amino acid sequence ofanother polypeptide (or portion thereof, which is at least 10, 20 or 50amino acids in length) that is not the immunoglobulin. Theimmunoglobulin, or fragment thereof, may be covalently linked to theother polypeptide at the N-terminus of the constant domain. Such fusionpolypeptides may facilitate purification, increase half-life in vivo,and enhance the delivery of an antigen across an epithelial barrier tothe immune system.

The immunoglobulins of embodiments of the invention include analoguesand derivatives that are modified, such as by the covalent attachment ofany type of molecule as long as such covalent attachment does not impairspecific binding. For example without limitation, the derivatives andanalogues of the immunoglobulins include those that have been furthermodified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein. In examples, chemical modifications of the analogues andderivatives are carried out by known techniques including, but notlimited to, specific chemical cleavage, acetylation, and formylation. Inadditional examples, the analogues or derivatives contain one or morenon-classical amino acids.

In various embodiments, the sErbB3 antibodies described herein are usedin methods known in the art relating to the localization and activity ofthe polypeptides of embodiments of the invention. Examples of useinclude without limitation, imaging or radioimaging these polypeptides,measuring amounts thereof in appropriate biological samples, indiagnostic, prognostic, and theragnostic methods, and for radiotherapy.

The antibodies of embodiments of the invention can be produced by anymethod known in the art for the synthesis of antibodies, in particular,by chemical synthesis or by recombinant expression, and may be producedby recombinant expression techniques.

Recombinant expression of antibodies, or fragments, derivatives oranalogs thereof, requires construction of a nucleic acid that encodesthe antibody. If the nucleotide sequence of the antibody is known, anucleic acid sequence encoding the antibody may be assembled fromchemically synthesized oligonucleotides, as described for example inKutmeier et al. (1994, BioTechniques 17:242), which, briefly, involvesthe synthesis of overlapping oligonucleotides containing portions of thesequence encoding antibody, annealing and ligation of thoseoligonucleotides, and then amplification of the ligated oligonucleotidesby PCR.

Alternatively, the nucleic acid encoding the antibody is obtained bycloning the antibody. If a clone containing the nucleic acid encodingthe particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the antibody may beobtained from a suitable source (e.g., an antibody cDNA library, or cDNAlibrary generated from any tissue or cells expressing the antibody) byPCR amplification using synthetic primers hybridizable to the 3′ and 5′ends of the sequence or by cloning using an oligonucleotide probespecific for the particular gene sequence.

Once a nucleic acid encoding at least the variable domain of theantibody molecule is obtained, it may be introduced into a vectorcontaining the nucleotide sequence encoding the constant region of theantibody molecule. Vectors containing the complete light or heavy chainfor co-expression with the nucleic acid to allow the expression of acomplete antibody molecule are also available. Then, the nucleic acidencoding the antibody can be used to introduce the nucleotidesubstitution(s) or deletion(s) necessary to substitute or delete the oneor more variable region cysteine residues participating in an intrachaindisulphide bond with an amino acid residue that does not contain asulfhydryl group. Such modifications can be carried out by any methodknown in the art for the introduction of specific mutations or deletionsin a nucleotide sequence, for example, but not limited to, chemicalmutagenesis, in vitro site directed mutagenesis, or PCR based methods.

Once a nucleic acid encoding an antibody of the invention has beenobtained, the vector for the production of the antibody may be producedby recombinant DNA technology using techniques well known in the art.Such methods can be used to construct expression vectors containing anantibody molecule coding sequence and appropriate transcriptional andtranslational control signals. These methods include, for examplewithout limitation, in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. See, for example, thetechniques disclosed in Sambrook et al. (2001, Molecular Cloning, ALaboratory Manual, 3d Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.) and Ausubel et al. (Current Protocols in MolecularBiology, John Wiley & Sons, NY).

A variety of host-expression vector systems can be utilized to expressan antibody molecule of embodiments of the invention. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced in large quantity and subsequently purified,but also represent cells which may, when transformed or transfected withthe appropriate nucleotide coding sequences, express the antibodymolecule of the invention in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli or B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing antibody coding sequences; yeast(e.g., Saccharomyces or Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing the antibody coding sequences; plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing antibody coding sequences; or mammalian cell systems(e.g., COS, CHO, BHK, HEK 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such apolypeptide is to be produced for the generation of pharmaceuticalcompositions comprising an antibody molecule, vectors which direct theexpression of high levels of fusion polypeptide products that arereadily purified may be desirable. Such vectors include but are notlimited to the E. coli expression vector pUR278 (Ruther et al., 1983,EMBO J. 2:1791) in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion polypeptide is produced; pIN vectors (Inouye & Inouye,1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J.Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be usedto express foreign polypeptides as fusion polypeptides with glutathioneS-transferase (GST). In general, such fusion polypeptides are solubleand can easily be purified from lysed cells by adsorption and binding toa matrix of glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetgene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into nonessential regions (for example the polyhedrin gene)of the virus and placed under control of an AcNPV promoter (for examplethe polyhedrin promoter). In mammalian host cells, a number ofviral-based expression systems (e.g., an adenovirus expression system orthose described above) may be utilized.

As discussed above, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in a specific way. Such modifications (e.g., glycosylation) andprocessing (e.g., cleavage) of polypeptide products may be important forthe function of the polypeptide.

For long-term, high-yield production of recombinant antibodies, stableexpression is preferred. For example, cell lines that stably express anantibody of interest can be produced by transfecting the cells with anexpression vector comprising the nucleotide sequence of the antibody andthe nucleotide sequence of a selectable marker (e.g., neomycin orhygromycin), and selecting for expression of the selectable marker. Suchengineered cell lines may be particularly useful for screening andevaluation of agents that interact directly or indirectly with theantibody molecule.

The expression levels of the antibody molecule can be increased byvector amplification.

The host cell may be co-transfected with two expression vectors, thefirst vector encoding a heavy chain derived polypeptide and the secondvector encoding a light chain derived polypeptide. The two vectors maycontain identical selectable markers which enable equal expression ofheavy and light chain polypeptides. Alternatively, a single vector maybe used which encodes both heavy and light chain polypeptides. In suchsituations, the light chain is preferably placed before the heavy chainto avoid an excess of toxic free heavy chain (see, e.g. Proudfoot, 1986,Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). Thecoding sequences for the heavy and light chains comprise either cDNA orgenomic DNA.

Once the antibody molecule has been recombinantly expressed, it may bepurified by any method known in the art for purification of an antibodymolecule, for example without limitation, by chromatography (e.g., ionexchange chromatography, affinity chromatography such as with protein Aor specific antigen, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of polypeptides.

In another embodiment, antibodies of the invention or fragments thereofare conjugated to a diagnostic or therapeutic moiety. The antibodies areused for diagnosis or to determine the efficacy of a given treatmentregimen. Detection is facilitated by coupling the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive nuclides, positronemitting metals (for use in positron emission tomography), andnonradioactive paramagnetic metal ions. See generally U.S. Pat. No.4,741,900 for metal ions which can be conjugated to antibodies for useas diagnostics. Suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;suitable prosthetic groups include streptavidin, avidin and biotin;suitable fluorescent materials include umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride and phycoerythrin; suitable luminescentmaterials include luminol; suitable bioluminescent materials includeluciferase, luciferin, and aequorin; and suitable radioactive nuclidesinclude .sup.125I, .sup.131I, .sup.111In and .sup.99Tc.

The therapeutic moiety is not to be construed as limited to classicalchemical therapeutic agents. In an example, the moiety may be a proteinor polypeptide possessing a desired biological activity. Such proteinsmay include, for example, a toxin such as abrin, ricin A, pseudomonasexotoxin, or diphtheria toxin; a polypeptide such as tumour necrosisfactor, .alpha.-interferon, .beta.-interferon, nerve growth factor,platelet derived growth factor, tissue plasminogen activator, athrombotic agent or an anti-angiogenic agent, e.g., angiostatin orendostatin; or, a biological response modifier such as a lymphokine,interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6),granulocyte macrophage colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), nerve growth factor (NGF) or othergrowth factor.

Techniques for conjugating such therapeutic moieties to antibodies arewell known to those skilled in the art, see for example withoutlimitation, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

Alternatively, in another example, an antibody is conjugated to a secondantibody to form an antibody heteroconjugate as disclosed for example inU.S. Pat. No. 4,676,980.

An antibody with or without a therapeutic moiety conjugated to it can beused as a therapeutic that is administered alone or in combination withcytotoxic factor(s) and/or cytokine(s).

Screening

Embodiments of the invention include methods for identifying activeagents (e.g., chemical compounds, proteins, or peptides) that bind to ansErbB3 polypeptide of embodiments of the invention and/or have astimulatory or inhibitory effect on the expression or activity of ansErbB3 polypeptide of the invention. Examples of active agents, include,but are not limited to, nucleic acids (e.g., DNA and RNA),carbohydrates, lipids, proteins, peptides, peptidomimetics, agonists,antagonists, small molecules and other drugs. Active agents can beobtained using any of the numerous suitable approaches in combinatoriallibrary methods known in the art, including without limitation:biological libraries; spatially addressable parallel solid phase orsolution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds. See, for examples, methods disclosed inLam, 1997, Anticancer Drug Des. 12:145; U.S. Pat. No. 5,738,996; andU.S. Pat. No. 5,807,683.

Various techniques are known in the art for screening polypeptides thatinteract with a protein such as sErbB3. Examples of polypeptides includesynthetic peptides, small molecular weight peptides (e.g., linear orcyclic peptides) or generated mutant gene products. Techniques forscreening large gene libraries often include cloning the gene libraryinto replicable expression vectors, transforming appropriate cells withthe resulting library of vectors, and expressing the genes underconditions in which detection of a desired activity, assembly into atrimeric molecule, binding to natural ligands, e.g., a receptor orsubstrate, facilitates relatively easy isolation of the vector encodingthe gene whose product was detected. Examples include withoutlimitation: two hybrid (interaction trap) assays, display libraries inwhich the candidate peptides are displayed on the surface of a cell,plasmid, or viral particle, and the ability of particular cells or viralparticles to bind an appropriate receptor protein via the displayedproduct is detected in a “panning assay”. In an example, such highthrough-put assays are followed or substituted by secondary screens,such as binding assays, to determine biological activities anddifferentiate agonists from antagonists.

Therapeutics and Methods of Treating

Methods and therapeutics to regulate sErbB3 in cancers, such ascarcinomas and gliomas, with aberrant sErbB3 expression or activity orwhich are regulated by HRG signaling or ErbB activity are embodiedherein. Such cancers include without limitation esophageal, liver,colon, gastric, thyroid, head and neck, kidney, bladder, pancreatic,lung, skin, breast, ovarian, cervical, endometrial, prostate, brain,intestinal, or testicular.

Various embodiments of the present invention include methods andtherapeutics to treat cancer, including carcinomas and gliomas, using ansErbB3 agent to regulate an sErbB3 by any available means includingwithout limitation nucleic acids (e.g., DNA and RNA), carbohydrates,lipids, proteins, peptides, peptidomimetics, agonists, antagonists,small molecules and other drugs. For example, various embodiments of thepresent invention pertain to regulation of p85-sErbB3 and/or p45-sErbB3or other sErbB3 isoforms. For example, the methods and therapeutics ofthe present invention comprise administering the nucleic acid sequencesSEQ ID NO:1 or SEQ ID NO: 3, or a complementary sequence thereof, or theamino acid sequences SEQ ID NO: 2 or SEQ ID NO: 4. The sequences includefor example, without limitation and as more fully described supra, thecomplete sequence, fragments and variants, as well as antisenseoligonucleotides. Additional embodiments of the present inventioninclude methods to regulate heregulin or ErbB signaling activity throughregulation of an sErbB3 agent. Effective amounts of an sErbB3 or anagonist or antagonist of an sErbB3 (function or expression) can bedetermined, for example, by decreased or increased heregulin activity,respectively as well as by effects on other biological endpoints such asreceptors, downstream mediators, or biochemical targets such as cellsurvival, cell growth, or metastasis.

One embodiment of the present invention relates to a method of treatingcancer in a subject, such as a human. The method includes administeringto the subject an agent that regulates an sErbB3 (function orexpression), such as p85-sErbB3 or p45-sErbB3, in an amount sufficientto reduce or prevent carcinoma cell growth and/or metastasis. The sErbB3agent may be administered in an amount effective to regulate heregulinactivation of ErbB receptor signaling activity, as well as by effects onother biological endpoints such as receptors, downstream mediators, orbiochemical targets such as cell survival, cell growth, or metastasis.

In examples, the agent comprises any of the previously describedpolypeptides, nucleic acid sequences, or variants thereof or previouslydescribed instruments. Examples include without limitation an sErbB3polypeptide, or a functional fragment, variant or analog thereof havingan sErbB3 activity; a polypeptide agonist or antagonist of sErbB3 thatincreases or decreases respectively the activity of an sErbB3 or thebinding of an sErbB3 to a binding partner; a small molecule thatincreases or decreases expression of an sErbB3, for example by bindingto the promoter region of the ErbB3 gene; an antibody, for example anantibody that binds to and stabilizes or assists the binding of sErbB3to an sErbB3 binding partner or an antibody that inhibits binding to anddestabilizes the binding of sErbB3 to an sErbB3 binding partner; or anucleotide sequence encoding an sErbB3 polypeptide or functionalfragment or analog thereof. The agent and instruments described hereinalso may comprise any compositions or methods in molecular medicine ortherapeutic transfer of genetic material known to those skilled in theart such as those previously described above to achieve a therapeuticeffect.

In an embodiment, the amount of sErbB3 protein is increased by elevatedtranscriptional expression of the endogenous sErbB3 gene and translationof the sErbB3 isoform from its alternate mRNA or by increasing sErbB3mRNA stability. In another embodiment, transcription of the sErbB3 geneis increased for example by altering its regulatory sequence such as bythe addition of a positive regulatory element (such as an enhancer or aDNA-binding site for a transcriptional activator); the deletion of anegative regulatory element (such as a DNA-binding site for atranscriptional repressor); and/or replacement of the endogenousregulatory sequence, or elements therein, with that of another gene,thereby allowing the coding region of the ErbB3 gene to be transcribedmore efficiently. In other examples, the amount of sErbB3 protein isregulated post-transcriptionally by microRNAS or post-transcriptionalregulatory elements.

In an embodiment, the agent is a vector that includes a nucleic acidsequence encoding an sErbB3, preferably a human sErbB3, for example SEQID NO: 2 or SEQ ID NO:4. The vector can be any vector suitable fortransfer of genetic material such as those listed previously or that areknown in the art.

In other embodiments, a therapeutic treatment or composition comprisesessentially the agent.

The agent can be administered by any method known in the art, forexample by direct administration, e.g., injection, intravenous orintramuscular. In another example, the agent is delivered directly to anaffected tissue. The agent can be coupled to a second agent, for examplea delivery agent (e.g., an agent that protects the agent fromdegradation) or a targeting agent (e.g., for targeting to the cancer oraffected tissue or targeting to the inside of a cell). Targeting mayoccur by means of, for example, the gene delivery vehicle, cell-type ortissue-type expression due to the transcriptional regulatory sequencescontrolling expression of the receptor gene, or a combination thereof.

The agent, e.g., an sErbB3 nucleic acid molecule, polypeptide, fragmentsor analog, or modulators (e.g., organic compounds and antibodies) can beincorporated into pharmaceutical compositions suitable foradministration to a subject, for example a human. Such compositions mayinclude the agent and a pharmaceutically acceptable carrier. As usedherein the language “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances are known.Except insofar as any conventional media or agent is incompatible withthe active compound, such media can be used in the compositions of theinvention. Supplementary active compounds can also be incorporated intothe compositions.

Examples of suitable carriers, excipients, and diluents are lactose,dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, alginates,gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water,methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearateand mineral oil. The compositions may additionally include solubilizingagents, lubricating agents, wetting agents, sweeteners, colorants,odorants, salts (polypeptides of embodiments of the present inventionmay themselves be provided in the form of a pharmaceutically acceptablesalt), buffers, coating agents, antioxidants, flavoring agents,emulsifiers, preservatives and the like.

A pharmaceutical carrier for hydrophobic compounds of embodiments of theinvention is a co-solvent system comprising benzyl alcohol, a nonpolarsurfactant, a water-miscible organic polymer, and an aqueous phase. Theproportions of a co-solvent system may be varied considerably withoutdestroying its solubility and toxicity characteristics.

The pharmaceutical composition may be adapted for administration by anyappropriate route, for example by the oral (including buccal orsublingual), rectal, nasal, topical (including buccal, sublingual ortransdermal), vaginal or parenteral (including subcutaneous,intramuscular, intravenous or intradermal) route. Such compositions maybe prepared by any method known in the art of pharmacy, for example byadmixing the active ingredient with the carrier(s) or excipient(s) understerile conditions.

Solutions or suspensions used for parenteral application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents, suspending agents, or thickeningagents; antibacterial agents such as benzyl alcohol or methyl parabens;antifungal agents such as parabens or thimerosal; antioxidants such asascorbic acid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide. The parenteral composition can beenclosed in ampoules, disposable syringes or multiple dose vials made ofglass or plastic. The composition may be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carried, for example water for injections, immediately prior touse. Extemporaneous injection solutions and suspensions may be preparedfrom sterile powders, granules and tablets. Prolonged adsorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., an agent described herein) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions,methods of preparation may include vacuum drying and freeze-drying whichyields a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Pharmaceutical compositions comprised of an sErbB3 agent adapted fororal administration may be presented as discrete units such as capsulesor tablets; as powders or granules; as solutions, syrups or suspensions(in aqueous or non-aqueous liquids; or as edible foams or whips; or asemulsions). Suitable excipients for tablets or hard gelatine capsulesinclude lactose, maize starch or derivatives thereof, stearic acid orsalts thereof. Suitable excipients for use with soft gelatine capsulesinclude, for example, vegetable oils, waxes, fats, semi-solid, or liquidpolyols etc. For the preparation of solutions and syrups, excipientswhich may be used include, for example, water, polyols and sugars. Forthe preparation of suspensions, oils (e.g. vegetable oils) may be usedto provide oil-in-water or water-in-oil suspensions.

Pharmaceutical compositions comprised of the sErbB3 agent adapted fortransdermal administration may be presented as discrete patches intendedto remain in intimate contact with the epidermis of the recipient for aprolonged period of time. For example, the active ingredient may bedelivered from the patch by iontophoresis as generally described inPharmaceutical Research, 3(6):318 (1986).

Pharmaceutical compositions comprised of an sErbB3 agent adapted fortopical administration may be formulated as ointments, creams,suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosolsor oils. When formulated in an ointment, the active ingredient may beemployed with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredient may be formulated in a cream withan oil-in-water cream base or a water-in-oil base. Pharmaceuticalcompositions adapted for topical administration to the eye include eyedrops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent. Pharmaceuticalcompositions adapted for topical administration in the mouth includelozenges, pastilles and mouth washes.

Pharmaceutical compositions comprised of an sErbB3 agent adapted forrectal administration may be presented as suppositories or enemas.

Pharmaceutical compositions comprised of an sErbB3 agent adapted fornasal administration wherein the carrier is a solid include a coarsepowder having a particle size for example in the range of 20 to 500microns which is administered in the manner in which snuff is taken,i.e. by rapid inhalation through the nasal passage from a container ofthe powder held close up to the nose. Suitable compositions wherein thecarrier is a liquid, for administration as a nasal spray or as nasaldrops, include aqueous or oil solutions of the active ingredient.

Pharmaceutical compositions comprised of an sErbB3 agent adapted foradministration by inhalation include fine particle dusts or mists whichmay be generated by means of various types of metered dose pressurisedaerosols, nebulizers or insufflators.

Pharmaceutical compositions comprised of an sErbB3 agent adapted forvaginal administration may be presented as pessaries, tampons, creams,gels, pastes, foams or spray compositions.

Pharmaceutical compositions of dragee cores are provided with suitablecoatings. For this purpose, concentrated sugar solutions may be usedwhich may optionally contain gum arabic, talc, polyvinyl pyrrolidone,carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquersolutions, and suitable organic solvents or solvent mixtures. Dyestuffsor pigments may be added to the tablets or dragee coatings foridentification or to characterize different combinations of activecompound doses.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

The compositions described herein can be formulated so as to providequick, sustained or delayed release of the active ingredient afteradministration to a patient by employing any of the procedures wellknown in the art. In one example, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. Liposomal suspensions (for example liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

The pharmaceutical compositions of embodiments of the present inventionmay be manufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

Thus, in an additional embodiment, the present invention provides apharmaceutical composition comprising at least one agent of embodimentsof the invention, optionally together with one or more pharmaceuticallyacceptable excipients, carriers or diluents. The pharmaceuticalcompositions can be included in a container, pack, or dispenser togetherwith instructions for administration.

The pharmaceutical composition may be provided in unit dosage form, andmay generally be provided in a sealed container, and may be provided aspart of a kit. Such a kit may include instructions for use and aplurality of the unit dosage forms.

Dosages of the active agent of embodiments of the present invention canvary between wide limits, depending upon the disease or disorder to betreated, and the age, condition, and response of the individual to betreated. A physician will ultimately determine appropriate dosages to beused. This dosage may be repeated as often as appropriate. If sideeffects develop the amount and/or frequency of the dosage can be reducedin accordance with normal clinical practice.

Embodiments of the invention further provide quantitative diagnostic andpharmaceutical packs and kits comprising one or more containers filledwith one or more of the ingredients of the aforementioned compositionsof embodiments of the invention. Associated with such container(s) canbe a notice in the form prescribed by a governmental agency regulatingthe manufacture, use or sale of pharmaceuticals or biological products,reflecting approval by the agency of the manufacture, use or sale of theproduct for human administration. Informational material can be includedwhich is descriptive, instructional, marketing or other material thatrelates to the methods described herein and/or the use of the agent forthe methods described herein. The kit may contain separate containers,dividers or compartments for the composition and informational material.For example, the composition can be contained in a bottle, vial, orsyringe, and the informational material can be contained in a plasticsleeve or packet. In other embodiments, the separate elements of the kitare contained within a single, undivided container. For example, thecomposition is contained in a bottle, vial or syringe that has attachedthereto the informational material in the form of a label. In variousembodiments, the kit includes a plurality of individual containers, eachcontaining one or more unit dosage forms of the agent. For example, thekit includes a plurality of syringes, ampules, foil packets, or blisterpacks, each containing a single unit dose of the agent. The containersof the kits can be air tight and/or waterproof. The kit optionallyincludes a device suitable for administration of the composition.

In a further embodiment, the present invention provides a method for thetreatment of cancer in a subject, for example a human, which comprisesadministering to the subject a therapeutically effective amount of atleast one active agent of the invention. Such agent may be in the formof a pharmaceutical composition as described herein. The method mayinclude more than one sErbB3 agent, more than one sErbB3 isoform, aswell as additional therapeutics or combinations thereof.

The present invention has been described with reference to specificdetails of particular embodiments thereof. It is not intended that suchdetails be regarded as limitations upon the scope of the inventionexcept insofar as and to the extent that they are included in theaccompanying claims.

EXAMPLES

Conditioned Media from Cells Expressing p45-sErbB3 and p85-sErbB3Inhibit HRG Activation of ErbB3. p45-sErbB3 and p85-sErbB3 are naturallyoccurring secreted products of the ErbB3 gene (Lee and Maihle 1998).p45-sErbB3 contains the amino-terminal 310 amino acids of ErbB3 and twounique carboxy-terminal amino acid residues. p85-sErbB3 contains theamino-terminal 519 amino acids of ErbB3 and 24 unique carboxy-terminalamino acid residues (See FIG. 1). To examine whether p45-sErbB3 andp85-sErbB3 can modulate HRG receptor activation cells stably transfectedwith these corresponding cDNA clones were isolated. These cells secretep45-sErbB3 and p85-sErbB3 into the culture medium (See FIG. 2A). Theconditioned medium from these cells was used as the source of p45-sErbB3or p85-sErbB3 in a series of experiments described below.

To test the ability of p45-sErbB3 and p85-sErbB3 to modulate aspects ofHRG-mediated ErbB receptor activation a clonal derivative of the Ba/F3cell line expressing exogenous ErbB2 and ErbB3 was stimulated with HRGαEGF domain₁₇₇₋₂₄₁ (HRGα) and HRGβ 1 EGF domain 176-246 (HRGβ) in theabsence or presence of concentrated conditioned media containingp45-sErbB3 and p85-sErbB3. As shown in FIG. 2, HRGβ was at least 20-foldmore effective than HRGα in stimulating ErbB3 tyrosine phosphorylation.Conditioned media containing sErbB3 inhibited HRGα-stimulated ErbB3activation by 40% (p45-sErbB3) and 80% (p85-sErbB3) at 1 μg/ml HRGα, asdetermined by densitometric analysis. However, at a higher concentration(2 μg/ml), conditioned media containing p85-sErbB3 decreased activationby 30%, although inhibition by conditioned media containing p45-sErbB3was negligible (See FIG. 2A). In the presence of conditioned mediumcontaining either p45-sErbB3 or p85-sErbB3, ligand stimulation of ErbB3tyrosine phosphorylation was decreased by 60% and 90%, respectively, atboth 50 and 100 ng/ml HRGβ (See FIG. 2C). These data indicate thatp85-sErbB3 inhibited ErbB3 phosphorylation in response to both HRGα andHRGβ more effectively than p45-sErbB3, although the concentration ofp85-sErbB3 used in these studies was lower than that of p45-sErbB3 (FIG.2A).

Purification of p85-sErbB3. p85-sErbB3 was isolated from a concentratedconditioned medium of cells stably transfected with a cDNA clone R31Fencoding p85-sErbB3 and was purified in two steps. The first step waslectin affinity chromatography with a Con A column (Sigma). The boundp85-sErbB3 was washed with column buffer (10 mM Tris-HCl, pH 7.5, 150 mMNaCl, 1 mM MnCl₂, and 1 mM CaCl₂) and eluted using column buffercontaining 1 M α-methyl D-mannoside, then dialyzed against 20 mMTris-HCl, pH 7.5 overnight. The second step of purification wasaccomplished using a Mono Q® anion exchange chromatography column forresolution of proteins and peptides on an FPLC® system, i.e., amicroprocessor controlled, solvent delivery apparatus used inpurification of biologically active compounds column (Pharmacia). Thebound p85-sErbB3 was eluted from the column with 0-500 mM NaCl gradientcontaining 20 mM Tris-HCl, pH 7.5. Samples taken from each step weresubjected to SDS-PAGE in duplicate and analyzed by Coomassie stainingand by Western blot using anti-ErbB3 236 antibody recognizing theextracellular domain of the ErbB3 (Lee and Maihle 1998). The finalp85-sErbB3 pool was homogeneous on SDS-PAGE, and the identity of thepurified protein was confirmed by Western blot analysis. Purifiedpreparations of p85-sErbB3 were used in all subsequent experiments.

p85-sErbB3 Binds to HRG with High Affinity. Previous reports based theassignation of the subdomain boundaries for the ErbB3 extracellulardomain on the subdomain boundaries of EGFR (Lee and Maihle 1998) asdefined by the genomic structure of avian ErbB1 (Callaghan, Antczak etal. 1993). Accordingly, p85-sErbB3 is composed of subdomains I throughIII and includes the first 45 amino acids of subdomain IV (aa 1-519),and a unique twenty-four amino acid sequence at the carboxy-terminus.Binding studies using heregulins indicate that subdomains I and II arerequired for heregulin binding (Singer, Landgraf et al. 2001). On theother hand, for EGF binding to EGFR subdomains I and III are low andhigh affinity binding sites, respectively (Lax, Bellot et al. 1989).

Direct binding between p85-sErbB3 and radiolabeled HRGβ was examinedusing the chemical crosslinker BS³. As shown in FIG. 3A, a proteincomplex of 90 kDa was formed between p85-sErbB3 and ¹²⁵I-HRGβ. Formationof this complex could be inhibited by addition of excess cold HRGβ butnot by addition of excess insulin, indicating that p85-sErbB3 binding toHRGβ is specific and that purified preparations of p85-sErbB3 arebiologically active. An analysis of ¹²⁵I—HRGβ₁₇₇₋₂₄₄ binding toimmobilized p85-sErbB3 was then performed using an ErbB3-IgG homodimeras a positive control. As shown in FIG. 3, p85-sErbB3 binds toHRGβ₁₇₇₋₂₄₄ with a K_(D) of 3.0±0.2 nM. In comparison, ErbB3-IgG bindsto HRGβ₁₇₇₋₂₄₄ with a K_(D) of 4.7±0.2 nM. These results demonstratethat p85-sErbB3 binds to HRGβ₁₇₇₋₂₄₄ with an affinity similar to that ofthe extracellular domain of ErbB3. The results of these twocomplementary experimental approaches establish the use of p85-sErbB3 tobind to HRG with an affinity equivalent to the affinity of HRG for thefull-length extracellular domain of ErbB3.

p85-sErbB3 Inhibits Binding of HRG to Receptors on the Cell Surface.p85-sErbB3 effectively limits binding of heregulin to cell surfacereceptors in the breast carcinoma cell line T47D. This cell lineexpresses all four ErbB receptors at moderate levels. Cells wereincubated with varying concentrations of p85-sErbB3 in the presence of¹²⁵I-labeled HRGβ₁₇₇₋₂₄₄. Simultaneously, a separate group of cells wasincubated with ¹²⁵I-HRGβ₁₇₇₋₂₄₄ in the presence of varyingconcentrations of 2C4, a monoclonal antibody specific for the ErbB2extracellular domain (Lewis, Lofgren et al. 1996). As shown by theinhibition curves (See FIG. 4), p85-sErbB3 and 2C4 inhibit HRGβ₁₇₇₋₂₄₄binding to cell surface receptors with similar IC₅s values (0.45±0.03 nMand 0.55±0.03 nM, respectively) although the mechanism of inhibition bythese two molecules is distinct. Although 2C4 inhibits heregulin bindingto cell surface receptors by blocking ErbB2-ErbB3 heterodimerization viabinding to the ErbB2 extracellular domain (Fitzpatrick, Pisacane et al.1998), p85-sErbB3 inhibits receptor activation and heterodimerization,at least in part, by competing for heregulin binding to the cellsurface.

p85-sErbB3 Blocks HRG-Induced Activation of ErbB2, ErbB3, and ErbB4. Theability of p85-sErbB3 to modulate HRG-stimulated receptor activation inthe Ba/F3 (ErbB2+ErbB3) cell line was examined using purifiedp85-sErbB3. This allowed an analysis of the mechanism of p85-sErbB3mediated inhibition in a quantitative manner. As shown in FIG. 5, whenBa/F3 (ErbB2+ErbB3) cells were treated with p85-sErbB3 at a 10-foldmolar excess over HRGβ₁₋₂₄₁, ErbB3 phosphorylation levels were reducedto basal levels. A similar level of receptor inhibition also wasapparent when either a 2.5- or 5-fold molar excess of p85-sErbB3 wasused in these experiments. Exogenous addition of p85-sErbB3 alsoinhibited HRG-induced ErbB2 activation. p85-sErbB3 blocked HRGstimulation whether the cells were treated with the EGF-like domain ofHRG (HRGα or HRGβ), as shown in FIG. 2, or with the entire HRGβ₁₋₂₄₁growth factor (See FIG. 5), suggesting that inhibition by p85-sErbB3occurs, at least in part, through a direct interaction betweenp85-sErbB3 and the EGF-like domain of HRG. Cells treated with the sameconcentration of p85-sErbB3 but not stimulated with HRG did not exhibitaltered ErbB2 or ErbB3 tyrosine phosphorylation, or show any change inthe level of either ErbB2 or ErbB3 expression, suggesting thatp85-sErbB3 does not function as a “ligand” for these receptors.

To examine whether exogenous addition of p85-sErbB3 exerts the sameinhibitory effect on endogenously expressed ErbB receptors, and todetermine whether p85-sErbB3 could modulate other members of the EGFreceptor family, the activity of p85-sErbB3 in two breast carcinoma celllines, i.e., T47D and MCF7, was tested. As shown in FIG. 6A, addition ofp85-sErbB3 (at a 6-fold molar excess relative to HRGβ) inhibitedHRG-induced activation of ErbB2, ErbB3, and ErbB4 in both the T47D andMCF7 cell lines. In contrast, at least in these two cell lines whichexpress low EGFR levels, EGFR phosphorylation remained at basal level incells treated with HRGβ regardless of whether p85-sErbB3 was present ornot. Similarly, EGF-induced phosphorylation of EGFR or ErbB2, or, to alesser degree, phosphorylation of ErbB3, was not decreased byp85-sErbB3. These results demonstrate that inhibition by p85-sErbB3 isspecific for HRG-induced activation of ErbB2, ErbB3, and ErbB4.

It is notable that in T47D cells, a decrease in ErbB2, ErbB3, and ErbB4protein levels following HRG stimulation was observed. In MCF7 cells adecrease in ErbB3 levels also was apparent when HRG was added to theculture medium (See FIG. 6A). It has been reported that the polyclonalErbB3 antibody specific to the carboxy-terminal 17 aa used in this studypreferentially recognizes non-phosphorylated ErbB3 on Western blots(Vartanian, Goodearl et al. 1997). Thus, when T47D or MCF7 cells arestimulated with HRG, a significant fraction of ErbB3 is phosphorylated,and, therefore, undetectable with this particular ErbB3 antibody. Theanti-ErbB antibodies used in these experiments recognize thecarboxy-terminal 17 aa (ErbB3) and 18 aa (ErbB2 and ErbB4) sequences ofthese receptors. Each of these sequences contains one tyrosine residue.Immunoblot detection by the anti-ErbB2 and ErbB4 antibodies used in thisstudy, therefore, may reflect either the level of receptor expression orthe unphosphorylated fraction of these receptors.

p85-sErbB3 Inhibits Activation of Downstream Effectors of HRG.HRG-stimulated activation of ErbB2, ErbB3, and ErbB4 leads to activationof two major signal transduction pathways: the PI3K pathway and the MAPKpathway (Wallasch, Weiss et al. 1995). To test whether p85-sErbB3 couldinhibit activation of these two downstream effector pathways in T47Dcells, activation of MAPK and Akt was examined by analyzing thephosphorylation levels of these proteins, and the ability of p85phosphatidylinositide 3-kinase (“PI3K”) to interact with ErbB3 followingHRGβ treatment. In the presence of p85-sErbB3 (10-fold molar excessrelative to HRGβ), tyrosine phosphorylation of ErbB3 was reduced tobasal levels. In the same cell population, addition of exogenousp85-sErbB3 abrogated the phosphorylation of both MAPK and Akt asdetermined by Western blot analysis, and inhibited ErbB3's associationwith p85 PI3K (See FIG. 6B). These results further demonstrate thatp85-sErbB3 can inhibit the activation of ErbB2, ErbB3, and ErbB4, andthis inhibition affects the activation of downstream signaling moleculessuch as MAPK, Akt, and PI3K.

p85-sErbB3 Inhibits HRG-stimulated Cell Growth. Inhibition ofHRG-induced phosphorylation of ErbB receptors by p85-sErbB3 correlateswith the modulation of HRG's biological effects. Specifically, a cellgrowth assay using MCF7 cells stimulated with HRGβ was performed andshowed that, within the concentration range tested, growth of this cellline was dose-dependent (See FIG. 7). It was observed that at aconcentration of 0.4 nM HRGβ the cell growth rate was half of the rateobserved at saturating levels of HRGβ. In cell cultures grown in thepresence of 0.4 nM HRGβ and p85-sErbB3 (a 100-fold molar excess relativeto HRGβ), p85-sErbB3 inhibited cell growth by 75% and 90%, at densitiesof 5,000 and 8,000 cells/well, respectively, whereas the sameconcentration of p85-sErbB3 did not affect cell growth in the absence ofHRGβ (See FIG. 7). Thus, the present invention discloses the use ofp85-sErbB3 as a potent inhibitor of HRG-dependent breast carcinoma cellgrowth in vitro.

p85-sErbB3 antibodies. Soluble ErBb3 antibodies are directed to theunique C-terminal regions. Rabbit polyclonal antibodies specific forp85-sErbB3 were generated using a 23 aa polypeptide from the uniquecarboxy-terminal region (Ser Lys Gly Ser Gln Ser Arg Met Gly Gly Gly GlyAla Leu Gln Trp Asn Cys Ser Gly Gly Ile Gln) and methods known in theart. Two rabbits were used; 10 ml preimmunize serum were collected fromboth rabbits, and both rabbits elicited high titer antibody productionin response to immunization with the 23-mer carboxy-terminus of p85sErbB3 as determined using enzyme-linked immunosorbent assays [ELISA]using the immunizing peptide as substrate; data not shown. Crudeantiserum (IgG preps from whole serum) from both rabbits specificallydetect an 85 kDa apparent molecular weight protein, that comigrates withan p85 kDa species detected by an anti-ErbB3 (ECD) antibody (FIG. 8); Ineither anti-sErbB3 antibody shows significant cross reactivity withother proteins (including p185 ErbB3) as demonstrated by immunoblotanalysis. These same antibody preparations were used to stainapproximately 50 breast tumors, using either preimmune serum or peptidecompetition controls, labeling was specific for p85-sErbB3 in bothnormal and malignant breast tissues. Results in both normal andmalignant breast tissue indicate a moderate to high level of sErbB3expression, restricted mainly to parenchymal epithelial cells, with noevidence of stromal staining. Importantly, the specificity of thisstaining pattern has been demonstrated through the use of bothpre-immune and peptide competition controls (data not shown).

p85-sErbB3 expression in tissues. Immunohistochemistry analysis usingthe p85-sErbB3 specific antibody, and using both pre-immune and peptidecompetition controls, showed p85-sErbB3 expression in most tissues ororgans, including the esophagus, stomach, liver, gall bladder, smallbowel, ureter, colon, thyroid gland, tonsils, lymph nodes, spleen,thymus, skeletal muscle, bronchioles, heart (epicardium and myocardium),hippocampus, head and neck, kidney, bladder, pancreas, adrenal gland,lung, skin, breast, ovary, uterus (cervix and endometrium), fallopiantube, placenta, prostate gland, brain, intestine, or testis. (data notshown).

1. A method for regulating heregulin activity, said method comprisingadministering an effective amount of an sErbB3 agent to a subject inneed thereof, wherein the sErbB3 agent is a p85-sErbB3 or p45-sErbB3polypeptide, and wherein the effective amount downregulates heregulinactivity.
 2. The method of claim 1 wherein said polypeptide consists ofSEQ ID NO: 2 or SEQ ID NO: 4.